Fig 6.

Definition of a Helix-Loop-Helix/E-box related recognition motif in Site I. Protein/DNA interactions at Site 1 were examined in vitro by EMSAs using a radio-labeled DNA binding site probe (WTRUNX2-HLH) spanning both a Runx2 and a HLH consensus motif and a panel of unlabeled competitor oligonucleotides (see Supplementary Fig. S2). Control binding reaction with nuclear proteins from MC3T3 cells generates two complexes that self-compete with the unlabeled WT RUNX2-HLH oligonucleotide. The upper complex also competes with oligonucleotides containing wildtype (HLH CON), but not mutant HLH (mHLH CON and WTRUNX2-mHLH) consensus motifs, and is mediated by an HLH/E-box related DNA binding activity. The lower complex competes with oligonucleotides containing Runx2 binding motifs.